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The Actin-Related Protein BAF53 Is Essential for Chromosomal Subdomain Integrity.

Lee K, Kim JH, Kwon H - Mol. Cells (2015)

Bottom Line: Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains.In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure.These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Yongin 449-791, Korea.

ABSTRACT
A chromosome territory is composed of chromosomal subdomains. The internal structure of chromosomal subdomains provides a structural framework for many genomic activities such as replication and DNA repair, and thus is key to determining the basis of their mechanisms. However, the internal structure and regulating proteins of a chromosomal subdomain remains elusive. Previously, we showed that the chromosome territory expanded after BAF53 knockdown. Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains. We found that BAF53 knockdown led to the disintegration of histone H2B-GFP-visualized chromosomal subdomains and BrdU-labeled replication foci. In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure. These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

No MeSH data available.


Related in: MedlinePlus

Early replication foci are selectively disintegrated by BAF53 knockdown. (A) The scheme of the experimental procedure. NIH3T3 cells were arrested at the G1/S border by double-thymidine blockade. After release from the G1/S arrest, the cells were pulse labeled for 20 min with BrdU at early or late S phase. Next, the cells were split into two groups—the control group was mock-treated, and the BAF53 knockdown group was infected with lentivirus containing shRNA-BAF53. At 48 h after the transduction, the cells were fixed and immunostained with anti-BrdU antibody. (B) The expression level of BAF53 at 48 h after the transduction was determined by immunoblotting. (C) Confocal midsections of the NIH3T3 cells labeled with BrdU at early S-phase. Pixels above the assigned thresholds were counted as replication foci in the cells. One hundred cells were counted for each experiment. Histograms show the number of replication foci in the control and BAF53-knockdown cell nuclei (n = 3, mean ± SD). t-test; P < 0.001. (D) Confocal midsections of the NIH3T3 cells labeled with BrdU at late S-phase. Pixels above the assigned thresholds were counted as replication foci in the cells. Thirty cells were counted for each experiment. The histograms show the number of replication foci in the control and BAF53-knockdown cell nuclei (n = 3, mean ± SD). t-test; P < 0.01.
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f3-molce-38-9-789: Early replication foci are selectively disintegrated by BAF53 knockdown. (A) The scheme of the experimental procedure. NIH3T3 cells were arrested at the G1/S border by double-thymidine blockade. After release from the G1/S arrest, the cells were pulse labeled for 20 min with BrdU at early or late S phase. Next, the cells were split into two groups—the control group was mock-treated, and the BAF53 knockdown group was infected with lentivirus containing shRNA-BAF53. At 48 h after the transduction, the cells were fixed and immunostained with anti-BrdU antibody. (B) The expression level of BAF53 at 48 h after the transduction was determined by immunoblotting. (C) Confocal midsections of the NIH3T3 cells labeled with BrdU at early S-phase. Pixels above the assigned thresholds were counted as replication foci in the cells. One hundred cells were counted for each experiment. Histograms show the number of replication foci in the control and BAF53-knockdown cell nuclei (n = 3, mean ± SD). t-test; P < 0.001. (D) Confocal midsections of the NIH3T3 cells labeled with BrdU at late S-phase. Pixels above the assigned thresholds were counted as replication foci in the cells. Thirty cells were counted for each experiment. The histograms show the number of replication foci in the control and BAF53-knockdown cell nuclei (n = 3, mean ± SD). t-test; P < 0.01.

Mentions: NIH3T3 cells were labeled with BrdU at the early or late S-phase and were split into two groups (Fig. 3A). The control group was mock-treated, and the BAF53-knockdown group was infected with lentivirus containing shRNA-BAF53 (Fig. 3B). Cells were furthered incubated for 48 h and then fixed and immunostained with anti-BrdU antibody. The number of pixels with staining above the assigned threshold per cell was counted. BAF53 knockdown dramatically reduced the number of replication foci pre-labeled at early S-phase (Fig. 3C). Because the cells of the control group and BAF53-knockdown group were derived from the same cell population labeled with BrdU, the cells of the control group and BAF53-knockdown group should contain the same extent of BrdU. Considering this, this result strongly suggested that BAF53 knockdown leads to unfolding of BrdU-prelabeled stable units of chromosome structure, and this unfolding reduces the intensity of BrdU staining of replication foci below the assigned thresholds, which can no longer be recognized as foci.


The Actin-Related Protein BAF53 Is Essential for Chromosomal Subdomain Integrity.

Lee K, Kim JH, Kwon H - Mol. Cells (2015)

Early replication foci are selectively disintegrated by BAF53 knockdown. (A) The scheme of the experimental procedure. NIH3T3 cells were arrested at the G1/S border by double-thymidine blockade. After release from the G1/S arrest, the cells were pulse labeled for 20 min with BrdU at early or late S phase. Next, the cells were split into two groups—the control group was mock-treated, and the BAF53 knockdown group was infected with lentivirus containing shRNA-BAF53. At 48 h after the transduction, the cells were fixed and immunostained with anti-BrdU antibody. (B) The expression level of BAF53 at 48 h after the transduction was determined by immunoblotting. (C) Confocal midsections of the NIH3T3 cells labeled with BrdU at early S-phase. Pixels above the assigned thresholds were counted as replication foci in the cells. One hundred cells were counted for each experiment. Histograms show the number of replication foci in the control and BAF53-knockdown cell nuclei (n = 3, mean ± SD). t-test; P < 0.001. (D) Confocal midsections of the NIH3T3 cells labeled with BrdU at late S-phase. Pixels above the assigned thresholds were counted as replication foci in the cells. Thirty cells were counted for each experiment. The histograms show the number of replication foci in the control and BAF53-knockdown cell nuclei (n = 3, mean ± SD). t-test; P < 0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4588722&req=5

f3-molce-38-9-789: Early replication foci are selectively disintegrated by BAF53 knockdown. (A) The scheme of the experimental procedure. NIH3T3 cells were arrested at the G1/S border by double-thymidine blockade. After release from the G1/S arrest, the cells were pulse labeled for 20 min with BrdU at early or late S phase. Next, the cells were split into two groups—the control group was mock-treated, and the BAF53 knockdown group was infected with lentivirus containing shRNA-BAF53. At 48 h after the transduction, the cells were fixed and immunostained with anti-BrdU antibody. (B) The expression level of BAF53 at 48 h after the transduction was determined by immunoblotting. (C) Confocal midsections of the NIH3T3 cells labeled with BrdU at early S-phase. Pixels above the assigned thresholds were counted as replication foci in the cells. One hundred cells were counted for each experiment. Histograms show the number of replication foci in the control and BAF53-knockdown cell nuclei (n = 3, mean ± SD). t-test; P < 0.001. (D) Confocal midsections of the NIH3T3 cells labeled with BrdU at late S-phase. Pixels above the assigned thresholds were counted as replication foci in the cells. Thirty cells were counted for each experiment. The histograms show the number of replication foci in the control and BAF53-knockdown cell nuclei (n = 3, mean ± SD). t-test; P < 0.01.
Mentions: NIH3T3 cells were labeled with BrdU at the early or late S-phase and were split into two groups (Fig. 3A). The control group was mock-treated, and the BAF53-knockdown group was infected with lentivirus containing shRNA-BAF53 (Fig. 3B). Cells were furthered incubated for 48 h and then fixed and immunostained with anti-BrdU antibody. The number of pixels with staining above the assigned threshold per cell was counted. BAF53 knockdown dramatically reduced the number of replication foci pre-labeled at early S-phase (Fig. 3C). Because the cells of the control group and BAF53-knockdown group were derived from the same cell population labeled with BrdU, the cells of the control group and BAF53-knockdown group should contain the same extent of BrdU. Considering this, this result strongly suggested that BAF53 knockdown leads to unfolding of BrdU-prelabeled stable units of chromosome structure, and this unfolding reduces the intensity of BrdU staining of replication foci below the assigned thresholds, which can no longer be recognized as foci.

Bottom Line: Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains.In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure.These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Yongin 449-791, Korea.

ABSTRACT
A chromosome territory is composed of chromosomal subdomains. The internal structure of chromosomal subdomains provides a structural framework for many genomic activities such as replication and DNA repair, and thus is key to determining the basis of their mechanisms. However, the internal structure and regulating proteins of a chromosomal subdomain remains elusive. Previously, we showed that the chromosome territory expanded after BAF53 knockdown. Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains. We found that BAF53 knockdown led to the disintegration of histone H2B-GFP-visualized chromosomal subdomains and BrdU-labeled replication foci. In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure. These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

No MeSH data available.


Related in: MedlinePlus